KR20090110936A - Therapeutic application of Kazal-type serine protease inhibitors - Google Patents

Abstract

The subject of the present invention is, in the most general aspect, the therapeutic application of the Kazal-type serine protease inhibitor lnfestin or domains thereof or modified Kazal-type serine protease inhibitors based on lnfestin homologs, which prevent the formation and/or stabilization of three-dimensional arterial or venous thrombi by interfering with proteins involved in activation of the so-called intrinsic coagulation pathway. In particular the present invention relates to the use of said Kazal-type serine protease inhibitors or fragments thereof or modified Kazal-type serine protease inhibitors, in the treatment or prophylaxis of a condition or disorder related to arterial thrombus formation, i. e. stroke or myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and/or diseases linked to pathological kinin formation such as hypotonic shock, edema including hereditary angioedema, bacterial infections, arthritis, pancreatitis, or articular gout, Disseminated Intravasal Coagulation (DIC) and sepsis.

Description

Therapeutic application of Kazal-type serine protease inhibitors

The object of the present invention is, in the most general aspect, Kazal-, which prevents the formation and / or stabilization of three-dimensional arterial or venous thrombus by interfering with the proteins involved in the activation of the so-called intrinsic coagulation pathway. type) Serine Protease Inhibitor The therapeutic application of a modified casal-type serine protease inhibitor based on Infestin or its domain or infestin homologue. In particular, the present invention relates to arterial thrombus formation, such as stroke or myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and / or hypotonic shock, edema including hereditary angioedema, bacterial infections, arthritis, pancreatitis or joints The present invention relates to the use of the casal-type serine protease inhibitor or fragment or modified casal-type serine protease inhibitor in the treatment or prevention of diseases associated with pathological kinin formation such as gout, disseminated intravascular coagulation (DIC) and sepsis. .

Vascular wall damage leads to the sudden attachment and aggregation of blood platelets, followed by the formation of fibrin-containing thrombin that occludes the site of injury and the activation of the plasma coagulation system. This phenomenon is important in limiting post-traumatic blood loss but can also lead to ischemia and infarction of critical organs by occluding diseased blood vessels. In the waterfall model, blood coagulation proceeds by a series of reactions involving zymogen activation by limited proteolysis, which peaks in the production of thrombin, which converts plasma fibrinogen into fibrin and platelets Activate it. The collagen- or fibrin-attached platelets are then multiplied several times through the exposure of coagulating phospholipids (primarily phosphatidyl serine) and platelet receptors and coagulation factors on their outer surface, which propagates the assembly and activation of coagulation protease complexes. Direct interaction between them promotes thrombin production.

There are two centralized pathways for coagulation initiated by exogenous (vascular wall) or endogenous (blood-origin) components of the vascular system. The "exogenous" pathway is integral membrane protein tissue factor (TF) and plasma factor VII (FVII), which are essential coagulation factors that are absent on the luminal surface but are strongly expressed in the subcutaneous layer of blood vessels and are accessible or freed through tissue damage. ) Is initiated by a complex. TF expressed in circulating microvessels can also contribute to thrombus proliferation by maintaining thrombin production on the surface of activated platelets.

An "endogenous" or contact activation pathway is initiated when factor XII [FXII, Hageman factor] contacts a negatively charged surface in a reaction involving high molecular weight kininogen and plasma kallikrein. FXII may be activated by macromolecular components of non-physiological materials such as endothelial matrices such as glycosaminoglycans and collagen, sulfite, nucleotides and free or polymers or other soluble polycationics. One of the strongest contact activators is kaolin and its response is the mechanistic basis for major clinical coagulation trials, providing an activated partial thromboplastin time (aPTT) that measures coagulation power via the "endogenous" pathway. do. In the response propagated by platelets, activated FXII subsequently activates FXI with FXIa and then FXIa activates factor IX. Complexes of FVIIIa and FIXa (tenase complexes) already activated by trace amounts of FXa and / or thrombin subsequently activate FX (see FIG. 1, "left"). Despite its high efficacy in inducing blood coagulation in vitro, the (patho) physiological significance of the FXII-initiated endogenous coagulation pathway has been associated with bleeding complications due to the genetic deficiency of FXII and high molecular weight kininogen and plasma kallikrein. It is questioned by the fact that it is not related. Along with the observation that humans and mice lacking endogenous pathway components such as TF and FVII suffer severe bleeding, this leads to the current hypothesis that an exogenous cascade is required for the stop of bleeding in vivo. Mackman, N. 2004.Role of tissue factor in hemostasis, thrombosis, and vascular development. Arterioscler. Thromb. Vasc. Biol. 24, 1015-1022).

In pathological conditions, the coagulation cascade may be activated inappropriately, which then results in the formation of a haemostatic plug in the blood vessels. As a result, blood vessels are blocked and blood supply to distant organs is limited. This process is known as thromboembolism and is associated with high mortality. In addition, the use of artificial instruments in contact with blood is severely limited due to the activation of the endogenous coagulation cascade. Proper coagulation of the artificial surface may in some cases avoid this problem but in others it may weaken its function. Examples of such artificial instruments are hemodialyser, cardiopulmonary by-pass circuit, heart valves, vascular stents and in-dwelling catheter. When such an apparatus is used, an anticoagulant such as heparin is administered to prevent fibrin formation on the surface. However, some patients do not tolerate heparin, which can lead to heparin-induced low thrombocytopenia (HIT), resulting in platelet coagulation and life-threatening thrombosis. In addition, an inherent disadvantage of all anticoagulants used in the clinic is the increased risk of severe bleeding. Therefore, there is a strong need for the presence of a new type of anticoagulant that is not associated with these complications and can be used as an excellent therapeutic concept to prevent thrombosis without increasing the risk of bleeding or suffering patients (Renne T et al. 2005). Defective thrombus formation in mice lacking factor XII.J.Exp.Med. 202: 271-281).

WO2006 / 066878 proposes the use of antibodies against FXII / FXIIa or the use of inhibitors of FXII / FXIIa. As a potent inhibitor, antithrombin III (AT III), angiotensin converting enzyme inhibitor, C1 inhibitor, aprotinin, alpha-1 protease inhibitor, antipine ([(S) -1-carboxy-2-phenylethyl] -carbamoyl- L-Arg-L-Val-Arginal), Z-Pro-Pro-aldehyde-dimethyl acetate, DX88 (Dyax Inc., 300 Technology Square, Cambridge, MA 02139, USA), Williams A and Baird LG DX-88 and HAE: a developmental perspective.Transfus Apheresis Sci. 29: 255-258), inhibitors of prolyl oligopeptidase such as leupeptin, Fmoc-Ala-Pyr-CN, corn-trypsin inhibitors, Sweet pumpkin trypsin inhibitor-V and Hamadarin, including mutants of bovine pancreatic trypsin inhibitor, ecotin, yellowfin sole anticoagulant protein, Cucurbita maxima isosuppressant (Isawa H et al. 2002. A mosquito salivary protein inhibits activation of the plasma contact system by binding to factor XII and high molecular weight kininoge n. as described in J. Biol. Chem. 277: 27651-27658).

An ideal inhibitor of FXII / FXIIa as a therapeutic agent with high inhibitory activity against FXII / FXIIa would not increase the risk of bleeding and would be non-immunogenic and ideally should be administered only rarely once. Small molecule inhibitors, such as Z-Pro-Pro-aldehyde-dimethyl acetate, have only a very short half-life or have been developed orally available sustained release after administration of the required multiple injections and should also be given constant over a long period of time. Human plasma proteins, such as C1 inhibitors, must first meet all requirements and have a relatively high inhibitory activity against FXII / FXIIa while not increasing the risk of bleeding and can be non-immunogenic as human proteins and also have a fairly long plasma half-life. have.

It has now been found that in vivo models of thrombogenic C1 inhibitors as key candidates for human FXII / FXIIa inhibitors cannot be successfully used to prevent occlusion. Other proposed FXII / FXIIa inhibitors from human plasma, ie AT III inhibitors, at least do not meet the second requirement, because using them as inhibitors of FXII / FXIIa may increase the risk of bleeding (see Warren BL et al. 2001. Caring for the critically ill patient.High-dose antithrombin III in severe sepsis: a randomized controlled trial.JAMA 286: 1869-1878).

Therefore, it is clear that there is still a need for improved medications for the treatment and / or prevention of thrombosis and similar diseases. Accordingly, it is an object of the present invention to satisfy this need.

It is known that deficiency of coagulation factor XII for more than 50 years is not associated with increased transient or damage-related bleeding complications (Ratnoff OD & Colopy JE 1955. A familial hemorrhagic trait associated with a deficiency of a clot-promoting fraction). of plasma.J Clin Invest 34: 602-613). Indeed, even if easily detected by the pathological value measured in aPTT (a clinical coagulation test focusing on the endogenous pathway of coagulation), a person lacking FXII does not cause abnormal bleeding even during major surgical operations [ See Colman RW. Hemostasis and Thrombosis. Basic principles & clinical practice (eds. Colman RW, Hirsch J, Mader VJ, Clowes AW, & George J) 103-122 (Lippincott Williams & Wilkins, Philadelphia, 2001)]. In contrast, deficiency of FXII is associated with an increased risk of venous thrombosis (Kuhli C et al. 2004. Factor XII deficiency: a thrombophilic risk factor for retinal vein occlusion. Am. J. Ophthalmol. 137: 459- 464; Halbmayer WM et al. 1993. Factor XII (Hageman factor) deficiency: a risk factor for development of thromboembolism. Incidence of FXII deficiency in patients after recurrent venous or arterial thromboembolism and myocardial infarction.Wien.Med.Wochenschr. 143: 43 -50). Supporting studies and case reports refer to John Hageman's case of FXII deficiency who died from pulmonary embolism. The hypothesis that FXII deficiency is associated with increased thrombotic risk is challenged by recent reassessment of some cases reporting the original report that FXII deficiency is associated with thrombosis (see Girolami A et al. 2004.The occasional venous thromboses seen in patients with severe (homozygous) FXII deficiency are probably due to associated risk factors: A study of prevalence in 21 patients and review of the literature.J. Thromb. Thrombolysis 17: 139-143). In most cases, the authors identified a factor FXII deficiency with concurrent innate or acquired thrombotic risk factors that could be involved in thrombosis phenomena independent of FXII. Well-characterized patients (Koster T et al. 1994. John Hageman's factor and deep-vein thrombosis: Leiden thrombophilia Study.Br. J. Haematol. 87: 422-424) and FXII-deficient households (Zeerleder S et. al. 1999.Reevaluation of the incidence of thromboembolic complications in congenital factor XII deficiency-a study on 73 subjects from 14 Swiss families.Thromb. Haemost. 82: 1240-1246). There is no association with sexual or anti-thrombotic risk.

Surprisingly and in contrast to the general idea of one skilled in the art, it has been found that Factor XII-derived endogenous coagulation pathways are involved in arterial thrombus formation in vivo but are not essential for normal tissue-specific hemostasis (Kleinschnitz). C et al. 2006. Targeting coagulation factor XII provides protection from pathological thrombosis in cerebral ischemia without interfering with hemostasis. J. Exp. Med. 203: 513-518; WO 2006/066878). Unexpectedly, these results put the factor XII in a central position in the course of pathological thrombus formation (FIG. 1). Thus, agents that can interfere with and block FXII activation or FXIIa activation may be suitable for blocking pathological arterial thrombus formation and its clinical outcome.

Recently, new inhibitors of FXII / FXIIa have been found in insects: Triatoma parasite, a vampire insect infestans ) Infestin domain 3-4 (Infestin 3-4) and Infestin domain 4 (Infestin-4) from mesentery (Campos ITN et al. 2002. Infestin, a thrombin inhibitor present in Triatoma infestans midgut , a Chagas' disease vector: gene cloning, expression and characterization of the inhibitor.Insect Biochem.Mol. Biol. 32: 991-997; Campos ITN et al. 2004. Identification and characterization of a novel factor XIIa inhibitor in the hematophagous insect , Triatoma infestans (Hemiptera: Reduviidae) .FEBS Lett. 577: 512-516). These proteins are known as potent FXIIa inhibitors of casal-type serine protease inhibitors that extend approximately 3-fold activated partial thromboplastin time.

These inhibitors have not been evaluated in terms of therapeutic applications that block pathological thrombus formation. In addition, this has not been attempted to reduce the immunogenicity of these heterologous inhibitors in humans and prolong their half-life in vivo.

Surprisingly it was found that the casal-type serine protease inhibitor domain Infestin-4 protects mice against pathological thrombus formation while no increased bleeding risk was observed in these animals. To increase plasma half-life, infestin-4 was expressed as a fusion to human albumin in mammalian cells and purified from cell culture supernatants. Purified inhibitors were injected into mice and a thrombotic challenge was induced with the aid of FeCl ₃ . 100% of mice treated with rHA-infestin-4 were protected, while vascular occlusion occurred in almost the majority of untreated control mice. The lack of associated bleeding risk is evidenced by a tail clipping experiment. Infestin-4 treated mice and untreated control mice show comparable haemostatic time and blood loss. That is, in vivo protection against thrombosis with negligible bleeding risk is demonstrated in mice against recombinant infestin-4. In this regard, Infestin 3-4 is included in terms of Infestin-4 but preferred compounds are mixtures of both Infestin-4 or Infestin 3-4 and predominantly Infestin-4.

rHA-Infestin-4 is also in vitro for its efficacy of specifically inhibiting endogenous pathways by measuring extended partial activated thromboplastin time (aPTT) in line with substances that effectively inhibit the endogenous pathway. Tested at In contrast, prothrombin time (PT), which is a test for factor VIIa / tissue factor-initiated activation of the exogenous pathway of coagulation, was little affected. Reduction of FXII activity was also demonstrated directly based on FXII deficient human plasma. Accordingly, an object of the present invention is a medicament, more specifically thrombosis, which prevents the formation and / or stabilization of three-dimensional arterial or venous thrombus without the risk of simultaneous bleeding by prolonging aPTT (PT is not necessarily affected). Use of the kazal-type serine protease inhibitor infestin or fragment thereof, preferably domain 3-4, most preferably domain 4, or fragment thereof, for the manufacture of a medicament for disease. Each inhibitor can thereby act to inhibit the activity of endogenous coagulation pathways, in particular FXIIa, and to inhibit the formation and / or stabilization of three-dimensional arterial or venous thrombi.

Accordingly, the present invention also provides respective pharmaceutical substances for the treatment or prevention of conditions or diseases associated with arterial thrombus formation, such as stroke or myocardial infarction. Due to the multiple effector actions of FXIIa, each drug substance can also be complement activated, fibrinolytic, inflammation, angiogenic and / or hypotonic shock, edema including hereditary angioedema, bacterial infections, arthritis, pancreatitis or joint gout, It has an additional therapeutic effect in diseases associated with pathological kinin formation, such as disseminated intravascular coagulation (DIC) and sepsis.

Deformed Kajal -Type serine protease inhibitors

Therapeutic administration of heterologous inhibitors such as infestin-4 in humans can produce an immune response. Thus, another object of the present invention is to identify an immunogenic but still potent casal-type serine protease inhibitor. Surprisingly by modifying what is associated with human casal-type serine protease inhibitors (serine protease inhibitor casal-1, SPINK-1) in such a way as to replace the presumed enzymatic contact site (s) with the corresponding regions of infestin-4. In particular, highly active FXIIa inhibitors have been produced that can be used in the manufacture of materials for treating or preventing thrombosis. Based on these results, it is possible to modify any natural kazal-type serine protease inhibitor in a manner that makes it FXIIa specific. An example is described in the following paragraphs.

In order to produce potent FXIIa inhibitors for therapeutic use in humans, we investigated human proteins with high similarity to infestin-4, which is not immunogenic in human patients than insect origin proteins. Human proteins with high similarity to infestin-4 were found to be SPINK-1, a casal-type serine protease inhibitor (also known as pancreatic secreted trypsin inhibitor, PSTI) expressed in the pancreas. The similarity between Infestin-4 and SPINK-1 is summarized in FIG. 2.

Based on the wild type SPINK-1 sequence, different mutants produced high similarity of the SPINK-1 sequence to infestin-4. Since there is no structural data for infestin, data for related inhibitors from Rhodnius prolixus in the complex with thrombin (PDB: 1TBQ) were analyzed. R. prolixus inhibitors have two kazal domains, the N-terminus of which interacts with the catalytic residues of thrombin. Thus, the N-terminal domain was used as a reference for comparison with Infestin-4. 3 shows the contact site of an R. phloxus inhibitor with thrombin and the contact site of SPINK-1 with chymotrypsin. A common feature of casal-type serine protease inhibitors is the accumulation of contact sites in the N-terminal region. Assuming that this region carries inhibitory specificity, several mutants of SPINK-1 have been generated. For the first mutant named K1, the predicted protease contact site in the amino-terminal site of SPINK-1 was replaced with that of infestin-4. Amino acid exchange in mutants K2 and K3 also changed SPINK-1 closer to the infestin-4 sequence. 4 shows the changes for the amino acid sequence and the SPINK-1 wild type sequence of these mutants. The amino acid sequences of the mature SPINK-1 wild type protein, three mutants and infestin-4 are shown in SEQ ID NOs: 1-5. The term "SPINK-1 mutant with increased increased similarity for each" means a mutant having at least 20 identical amino acids with infestin-4, or a mutant with conservative substitutions instead of identity meaning conservative substitutions other than the same amino acid. . These mutants are different from the mutants of human pancreatic secretory trypsin inhibitors described in EP-A2-0352089, WO88 / 03171A and EP-A2-0278112.

SPINK-1 mutants were expressed and purified. Mutants are tested for their potential to specifically inhibit endogenous pathways by measuring extended partial thromboplastin time (aPTT), as predicted in the case of substances capable of inhibiting endogenous pathways. It was tested in vitro. In contrast, prothrombin time (PT), a test for factor VIIa / tissue factor-initiated activation of the exogenous pathway of coagulation, was not necessarily affected.

Accordingly, another aspect of the invention is a medicament, more particularly a mammalian kazal-type serine protease inhibitor SPINK-1, infestin homolog or fragment thereof, or preferably infestin domain 4, for the manufacture of a medicament for thrombosis. Or modified forms of fragments and homologues thereof. The mode of action of this symptom is the inhibition of FXII / FXIIa activity, which can be measured by prolonged (PT is not necessarily affected) aPTT. In this way, the formation and / or stabilization of three-dimensional arterial or venous thrombus is prevented. Each inhibitor may act as an inhibitor of the activity of endogenous coagulation pathways, in particular FXIIa, as long as it inhibits the formation and / or stabilization of three-dimensional arterial or venous thrombi.

Accordingly, the present invention also relates to conditions or diseases associated with arterial thrombus formation, such as stroke or myocardial infarction, complement activation, fibrinolysis, inflammation, angiogenesis and / or hypotonic shock, edema including hereditary angioedema, bacterial infection , Risk of immunogenicity in humans for the treatment or prevention of diseases associated with pathological kinin formation, such as cancer (Trousseau syndrome), arthritis, pancreatitis or joint gout, disseminated intravascular coagulation (DIC) or sepsis To provide this reduced material.

Another particularly preferred protein of the invention is the human pancreatic protease inhibitor SPINK-1 induced mutant as described above, wherein the mutant is associated with the infestin-4 protein in relation to their ability to inhibit FXIIa activity. It is characterized by a change to increase their similarity. Such mutants are characterized in that they prolong the partial thromboplastin time activated in vitro.

Modified Mammals with Extended Half-Life Kajal -Type serine protease inhibitors Infestin -4 and modified Casal  Serine Protease Inhibitors

Another aspect of the invention is a modified mammalian kazal-type serine protease inhibitor based on infestin-4 and infestin homolog or fragment thereof having extended half-life. Since the Kazal-type serine protease of the present invention is somewhat bovine protein, it is possible to predict a rapid kidney clearance rate as published for other bovine proteins (Werle M. and Bernkop-Schnurch A. 2006. Strategies to improve plasma half). -life time of peptide and protein drugs.Amino Acids 30: 351-367). One way to solve the short plasma half-life of the polypeptide compounds is of course to inject them either repeatedly or via continuous infusion. Preferably the endogenous plasma half-life of the polypeptide itself is increased. Accordingly, another aspect of the present invention is to provide a casal-type serine protease inhibitor fused to a half-life extending protein (HLEP).

As used herein, a “half-life enhancing polypeptide” (HLEP) refers to albumin, a member of the albumin-family, a constant region of immunoglobulin G and fragments thereof and sites of albumin, a member of the albumin family and an immunoglobulin constant region under physiological conditions. It is selected from the group consisting of polypeptides that can bind.

Albumin and immunoglobulins and fragments or derivatives thereof are described as specific examples of half-life enhancing polypeptides (HLEP).

Balance et al., In WO 01/79271, refer to fusion polypeptides of many different therapeutic polypeptides that are predicted to have increased functional half-life and extended shelf-life in vivo when fused to human serum albumin. Described. Therapeutic proteins can be fused to albumin residues either directly or via peptidic linkers, with C- and N-terminal fusions described.

The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are more broad and include human serum albumin (and fragments and variants thereof) and albumin (and fragments and variants thereof) from other species.

As used herein, "albumin" refers to an albumin polypeptide or amino acid sequence, or albumin fragment or variant, collectively having one or more functional activities (eg, biological activity) of albumin. In particular, "albumin" refers to human albumin or fragments thereof, especially albumin or fragments thereof, or analogs or variants or fragments thereof, of mature forms of human albumin or from other vertebrates as shown in SEQ ID NO: 6 herein.

The albumin moiety of the albumin fusion protein may comprise a full length HA sequence as described above, or may comprise one or more fragments thereof that may stabilize or prolong the therapeutic activity. Such fragments may be 10 or more amino acids in length or may comprise about 15, 20, 25, 30, 50 or more contiguous amino acids from the HA sequence or may include some or all of the specific domains of HA.

The albumin moiety of the albumin fusion protein of the invention may be a variant of normal HA. The therapeutic polypeptide moiety of the fusion protein of the invention may also be a variant of the corresponding therapeutic polypeptide as described herein. The term “variant” includes conservative or non-conservative insertions, deletions and substitutions, wherein such changes do not substantially alter the active site or active domain conferring the therapeutic activity of the therapeutic polypeptide.

In particular, albumin fusion proteins of the invention may include naturally occurring polymorphic variants of human albumin and fragments of human albumin. Albumin may originate from any vertebrate, especially any mammal, eg, human, monkey, cow, sheep or pig. Non-mammal albumin includes, but is not limited to, those derived from hens and salmon. The albumin moiety of the albumin-linked polypeptide may be from different animals besides the therapeutic polypeptide moiety.

In general, albumin fragments or variants will be at least 20, preferably at least 40, most preferably at least 70 amino acids in length. Albumin variants are preferably at least one full domain of albumin or fragments of such domains, such as domain 1 (amino acids 1-194 of SEQ ID NO: 6), 2 (amino acids 195-387 of SEQ ID NO: 6), 3 ( Amino acids 388-585 of SEQ ID NO: 6, 1 + 2 (amino acids 1-387 of SEQ ID NO: 6), 2 + 3 (amino acids 195-585 of SEQ ID NO: 6) or 1 + 3 (amino acids 1-194 of SEQ ID NO: 6) + Amino acids 388-585 of SEQ ID NO: 6) or alternatively. Each domain itself has two homologous subdomains, 1-105, 120-194, having a flexible inter-subdomain linker region comprising residues Lys106 to Glu119, Glu292 to Val315, and Glu492 to Ala511. 195-291, 316-387, 388-491 and 512-585.

The albumin moiety of the albumin fusion protein of the invention may comprise at least one subdomain or domain of HA or a conservative modification thereof. In addition to albumin, another member of the albumin family, alpha-fetoprotein, is claimed to extend the half-life of therapeutic polypeptides attached in vivo (WO 2005/024044). Albumin family of proteins that are evolutionarily related to serum transfer proteins are albumin, alpha-fetoprotein (AFP; see Beattie & Dugaiczyk 1982.Structure and evolution of human alpha-fetoprotein deduced from partial sequence of cloned cDNA.Gen. 20: 415- 422), alphamin (AFM; see Lichenstein et al. 1994. Afamin is a new member of the albumin, alpha-fetoprotein, and vitamin D-binding protein gene family.J. Biol. Chem. 269: 18149-18154) And vitamin D binding protein (DBP; Cooke & David 1985. Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family.J. Clin. Invest. 76: 2420-2424). Their genes represent multigenic clusters with structural and functional similarities that map to the same chromosomal region in humans, mice and rats. Structural similarity of albumin family members suggests their utility as HLEP. Accordingly, another object of the present invention is to use such albumin family members, fragments and variants thereof as HLEPs. The term “variant” includes conservative or non-conservative insertions, deletions and substitutions, wherein such changes do not substantially alter the active site or active domain conferring the therapeutic activity of the therapeutic polypeptide.

Albumin family members may comprise each protein AFP, AFM and DBP of full length, or may comprise one or more fragments thereof that may stabilize or prolong the therapeutic activity. Such fragments may be at least 10 amino acids in length or may comprise at least about 15, 20, 25, 30, 50 or more contiguous amino acids of each protein sequence or may comprise some or all of the specific domains of each protein. Can be.

Albumin family member fusion proteins of the invention may include naturally occurring polymorphic variants of AFP, AFM and DBP. The protein may be from any vertebrate, in particular from any mammal, for example human, monkey, cow, sheep or pig. Non-mammal albumin family members include, but are not limited to, those derived from hens and salmon.

IgG and IgG-fragments without antigen-binding domains can also be used as HLEPs. The therapeutic polypeptide site is preferably linked to the IgG or IgG fragment via the hinge region of the peptidic linker or antibody, which may be cleaved. Several patents and patent applications describe the fusion of therapeutic proteins to immunoglobulin constant regions to extend their half-life in vivo. US 2004/0087778 and WO 2005/001025 describe fusion proteins of at least a portion of a biologically active peptide and immunoglobulin constant region or Fc domain which increase the half-life of the peptide and also rapidly degrade in vivo. have. The Fc-IFN-β fusion protein described increased biological activity, extended circulating half-life, and higher solubility (WO 2006/000448). Fc-EPO protein with extended serum half-life and increased in vivo efficacy (WO 2005/063808) and Fc fusion with G-CSF (WO 2003/076567), glucagon-like peptide-1 (WO 2005/000892), coagulation factor (WO 2004/101740) and interleukin-10 (see US 6,403,077) are described, all of which have half-life properties.

Accordingly, another aspect of the invention relates to the use of such immunoglobulin sequences, preferably Fc fragments and variants thereof as HLEP. Casal-type serine protease inhibitors, such as infestin-4, and modified casal-type serine protease inhibitors with improved inhibitory specificity for FXIIa, such as SPINK-1 mutants, may be present in at least a portion of an immunoglobulin constant region, such as the Fc domain or HLEP. It can be fused and expressed in E. coli , yeast, insect, plant or vertebrate cells or transgenic animals. The SPINK-K2-Fc fusion protein is exemplarily shown in SEQ ID NO: 25.

The present invention specifically relates to a casal-type serine protease inhibitor such as infestin-4 and a modified casal-type serine protease inhibitor such as a SPINK-1 mutant or fragment or variant thereof, the N- or HLEP or fragment or variant thereof. Fusion proteins formed by linking to the C-terminus have an increased in vivo half-life compared to the corresponding Kazal-type serine protease inhibitors not linked to HLEP. Intervening peptidic linkers can be introduced between the therapeutic polypeptide and the HLEP. Cleavable linkers can be introduced when HLEP interferes with the specific activity of the therapeutic polypeptide, for example by steric hindrance. Preferred enzymes for linker cleavage are coagulation proteases of the endogenous coagulation pathway, FXIIa, FXIa, FIXa, FVIIIa or FXa, wherein the most preferred cleavage enzyme is FXIIa.

The casal-type serine protease inhibitor family is one of a number of families of serine protease inhibitors. Many proteins from different species have been described (Laskowski M and Kato I. 1980. Protein inhibitors of proteinases. Ann. Rev. Biochem. 49: 593-626).

"Infestin-4 and modified casal-type serine protease inhibitors" in the above definitions encompass polypeptides having a natural amino acid sequence or SEQ ID NOs: 2-5 or 21-24. However, this definition also includes a modified N-terminus comprising a slightly modified amino acid sequence, eg, terminal amino acid deletions or additions, so long as such polypeptides substantially retain the activity of each casal-type serine protease inhibitor. Or a polypeptide having a C-terminal end. "Casal-type serine protease inhibitors" in the above definitions include naturally occurring allelic variations that may each occur individually and may occur. "Casal-type serine protease inhibitors" in the above definitions also include variants of casal-type serine protease inhibitors. Such variants differ in at least one amino acid residue from the wild type sequence. Examples of such differences include truncation of the N- and / or C-terminus by one or more amino acid residues (eg, 1 to 10 amino acid residues), or one or more additional at the N- and / or C-terminus. Addition of residues, and conservative amino acid substitutions, eg, amino acids having similar properties, such as (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5 A) hydrophobic amino acids, and (6) substitutions within the group of aromatic amino acids. Examples of such conservative substitutions are shown in Table 1.

(One)  Alanine glycine (2)  Aspartic Acid Glutamic Acid (3a)  Asparagine Glutamine (3b)  Serine Threonine (4)  Arginine Histidine Lysine (5)  Isoleucine Leucine Methionine Valine (6)  Phenylalanine Tyrosine Tryptophan

The present invention also relates to polynucleotides encoding a casal-type serine protease inhibitor as described herein. The term “polynucleotide (s)” generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. The polynucleotides may be single-stranded or double stranded DNA, single stranded or double stranded RNA. The term "polynucleotide (s)" as used herein also includes DNA or RNA comprising an unusual base such as inosine and / or one or more modified bases. It will be appreciated that various modifications may be made to DNA and RNA that serve many useful purposes known to those skilled in the art. As used herein, the term “polynucleotide (s)” refers to polynucleotides in such chemically, enzymatically or metabolically modified forms, and viruses and cells, including, for example, mononuclear and complex cells. And chemical forms of RNA.

The skilled artisan will understand that due to the degeneracy of the genetic code, the provided polypeptides can be encoded by different polynucleotides. These "variants" are encompassed by the present invention.

Preferably, the polynucleotides of the present invention are isolated polynucleotides. The term “isolated” polynucleotides refers to polynucleotides that are substantially free from other nucleic acid sequences, such as, but not limited to, other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides can be purified from host cells. Isolated polynucleotides can be obtained using conventional nucleic acid purification methods known to the skilled person. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

Another aspect of the invention is a plasmid or vector comprising a polynucleotide according to the invention. Preferably the plasmid or vector is an expression vector. In certain embodiments, the vector is a delivery vector for use in human gene therapy.

Another aspect of the invention is a host cell comprising a polynucleotide or plasmid or vector of the invention.

The host cell of the invention can be used in a method of producing a casal-type serine protease inhibitor which is part of the invention. The method

Culturing the host cell of the invention under conditions in which a casal-type serine protease inhibitor is expressed; And

Optionally recovering a Kazal-type serine protease inhibitor from the culture medium.

Proposed Of polypeptide  Expression:

Casal-type serine proteases and modified Kazal-type serine protease inhibitors of the invention are produced as recombinant molecules in prokaryotic or eukaryotic host cells such as bacteria, yeasts, plants, animals (including insects) or human cell lines or transgenic animals. Can be.

Animals or people In cell lines  Expression

High levels of production of recombinant proteins in suitable host cells are suitable for recombinant expression vectors in which the above-mentioned modified cDNA can be propagated in various expression systems according to methods known to those skilled in the art in efficient transcription units. It requires assembly with the control component. Efficient transcriptional regulatory components may originate from viruses with animal cells as their natural host or from chromosomal DNA of animal cells. Preferably, it is potent in animal cells such as beta actin or GRP78, or promoter-enhancer combinations derived from Simian virus 40, adenovirus, BK polyoma virus, human cytomegalovirus, or long terminal repeat units of Raus sarcoma virus. Promoter-enhancer combinations can be used that comprise constitutively transcribed genes. To achieve stable high levels of mRNA transcribed from cDNA, the transcription unit must contain a DNA region encoding the transcriptional end-polyadenylation sequence at its 3'-adjacent site. Preferably, the sequence is from the Simian virus 40 initial transcription region, rabbit beta-globin gene, or human tissue plasminogen activator gene.

The cDNA is then transfected into a suitable host cell line for expression of the therapeutic polypeptide. Examples of cell lines that can be used are monkey COS-cells, mouse L-cells, mouse C127-cells, hamster BHK-21 cells, human embryonic kidney 293 cells and hamster CHO-cells.

Recombinant expression vectors encoding the corresponding cDNA can be introduced in several different ways. For example, recombinant expression vectors can be generated from vectors based on different animal viruses. Examples of these are vectors based on baculovirus, vaccinia virus, adenovirus and preferably bovine papilloba virus.

Transcriptional units encoding corresponding DNAs also contain animal cells along with other recombinant genes that can act as predominant selectable markers within these cells to facilitate the isolation of specific cell clones that integrate the recombinant DNA into their genomes. Can be introduced into. Examples of predominant selectable marker genes of this type include Tn5 amino glycoside phosphotransferase, which confers resistance to geneticin (G418), hygromycin phosphotransferase, which confers resistance to hygromycin, and Puromycin acetyl transferase that confers resistance to puromycin. Recombinant expression vectors encoding such selectable markers can be present on the same vector encoding the cDNA of the protein of interest, or they are encoded on separate vectors that are co-introduced and integrated into the genome of the host cell, often It is possible to create strong physical connections between different transcription units.

Another type of selectable marker gene that can be used with the cDNA of the desired protein is based on various transcription units encoding dihydrofolate reductase (dhfr). This type of gene will be introduced into cells lacking endogenous dhfr-activity, preferably CHO-cells (DUKX-B11, DG-44) and then allowed to grow in nucleoside deficient media. Examples of such media are Ham's F12 free of hypoxanthine, thymidine and glycine. By introducing these dhfr-genes into the CHO-cells of this type linked on the same vector or on different vectors with the cDNA transcriptional units of the Kazal-type serine protease inhibitor, a dhfr-positive cell line producing recombinant protein will be produced. Can be.

If the cell line grows in the presence of the cytotoxic dhfr-inhibitor methotrexate, a new cell line will emerge that is resistant to methotrexate. These cell lines can produce recombinant proteins at an increased rate due to the amplified number of linked dhfr and the transcriptional unit of the desired protein. When propagating these cell lines in increasing concentrations of methotrexate (1-10000 nM), new cell lines can be obtained that produce very high ratios of the desired protein.

The cell line producing the desired protein can be grown on a large scale in suspension culture or various solid supports. Examples of such supports are microcarriers based on dextran or collagen matrices or solid supports in the form of hollow fibers or various ceramic materials. When growing in cell suspension culture or on a microcarrier, the cell line can be cultured as a batch culture or a perfusion culture in which a conditioned medium is continuously produced over an extended period of time. That is, according to the present invention, the cell line is very suitable for the development of industrial processes for the production of the desired recombinant protein.

Recombinant proteins that accumulate in a medium that secretes cells of this type include methods that utilize differences in size, charge, hydrophobicity, solubility, specific affinity, etc., between the desired protein and other materials in the cell culture medium, It can be concentrated or purified by various biochemical and chromatographic methods.

An example of such purification is the adsorption of recombinant proteins to monoclonal antibodies or binding peptides immobilized on a solid support. After adsorption, the protein can also be purified by various chromatographic techniques based on these properties.

Expression in the Yeast Expression System

Exemplary yeast genera considered to be useful as examples of the invention as hosts include Pichia , previously classified as Hansenula, Saccharomyces , Kluyveromyces , Aspergillus. a (Aspergillus), Candida (Candida), sat rulrop sheath (Torulopsis), torulra spokes La (Torulaspora), ski irradiation Caro My process (Schizosaccharomyces), key interrogating my process (Citeromyces), Parkinson solren (Pachysolen), Eisai Kosaka Mai Zygosaccharomyces , Debaromyces , Tricho derma , Cephalosporium , Humicola , Mucor , Neurospora , Yarrow Yarrowia , Metschunikowia , Rhodosporidium , Leucosporidium , Botryoascus , Sporidiobolus , Endocorpsis Endomy copsis ). Genus includes those selected from the group consisting of Saccharomyces, Ski Research Caromyces, Kluiberomyces, Pichia and Torulaspora. Examples of Saccharomyces subspecies are S. cerevisiae, S. italicus and S. rouxii .

Suitable promoters for S. cerevisiae include PGKI gene, GAL1 or GAL10 gene, CYCI, PHO5, TRPI, ADHI, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phos A portion or upper activation site of the 5 'regulatory region of popructokinase, trios phosphate isomerase, phosphoglucose isomerase, glucokinase, alpha-crossing factor pheromone, PRBI, GUT2, GPDI promoter and other promoters (e.g. Eg, those related to genes for hybrid promoters, including hybrids of promoters EP-A-258 067) and portions of the 5 ′ regulatory region.

Convenient adjustable promoters for use in Schizosaccharomyces pombe are described in Maundrell, Maundrell K. 1990. Nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J. Biol. Chem. 265: 10857-10864, Thiamine-inhibitable promoters from the nmt gene as described in Hoffman and Winston, Hoffman CS and Winston F. 1990. Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe. Genetics 124: 807-816, a glucose inhibitable jbpI gene promoter.

The transcription termination signal may be the 3 'flanking sequence of the eukaryotic gene containing the appropriate signal for transcription termination and polyadenylation. Suitable 3 'flanking sequences can be, for example, those of genes naturally linked to the expression control sequences used, and can correspond, for example, to a promoter. Alternatively, they may be different if the termination signal of the ADHI gene is used optionally in S. cerevisiae.

Expression in Bacterial Expression Systems

Exemplary expression systems for the production of the modified casal-type serine protease inhibitors of the invention in bacteria are Bacillus subtilis ( Bacillus subtilis). subtilis), Bacillus brevis (Bacillus brevis ), Bacillus megaterium megaterium ), Caulobacter crescentus ) and most importantly Escherichia coli BL21 and E. coli K12 and derivatives thereof. Convenient promoters include trc promoter, tac promoter, lac promoter, lambda phage promoter p _L , L-arginose inducible araBAD promoter, L-rhamnose inducible rhaP promoter and anhydrotetracycline-induced tetA promoter / operator However, the present invention is not limited thereto.

In one embodiment, the polynucleotides encoding the infestin and modified casal-type serine protease inhibitors of the invention direct localization of the protein of the invention to specific compartments of the prokaryotic cell and / or from the prokaryotic cell Can be fused to a signal sequence that will direct the secretion of the protein. For example, in E. coli, it may be required to direct the expression of the protein into the periplasmic region. Examples of signal sequences or proteins (or fragments thereof) to which proteins of the proteins of the invention can be fused to direct polypeptides to be expressed in the bacterial cytoplasmic space include pelB signal sequences, maltose binding protein signal sequences, ompA signals. Sequences, peripheral cytoplasmic E. coli heat-labile enterotoxin B-subunits, and alkaline phosphatase signal sequences. Several vectors are available commercially for the construction of fusion proteins that will direct the localization of proteins such as the pMAL family of vectors (New England Biolabs).

Expression in plant cells

Exemplary plant systems for the expression of the modified casal-type serine protease inhibitors of the present invention include tobacco, potatoes, rice, corn, soybeans, alpha waves, tomatoes, lettuce and legumes. Ma JKC et al. 2003 The production of recombinant pharmaceutical proteins in plants.Nat. Rev. Genet. 4: 794-805). Expression of recombinant proteins in plant systems can be directed by regulatory elements suitable for specific organs or tissues, such as fruits, seeds, leaves or tubers. Alternatively, the protein can be secreted from the roots. In cells, proteins can be targeted to specific compartments, such as cytoplasmic reticulum, protein bodies or plastids. Here, the product may accumulate to higher levels or undergo certain forms of post-translational modification.

Transgenic expression

Illustrative examples of large-scale transgenic expression systems (Pollock DP. 1999. Transgenic milk as a method for the production of recombinant antibodies.J Immunol Methods 231: 147-157) are rabbits (Chrenek P et al. 2007). Expression of recombinant human factor VIII in milk of several generations of transgenic rabbits.Transgenic Res. 2007 Jan 31), Goat (Lazaris A et al. 2006. Transgenesis using nuclear transfer in goats.Methods Mol Biol. 348: 213- 26), pigs and cattle.

Tablets and Therapeutic Formulations

It is preferred to purify the casal-type serine protease inhibitors of the invention to at least 80% purity, more preferably at least 95% purity, at least 99.9% pure with respect to contaminating macromolecules, in particular with respect to other proteins and nucleic acids, Particularly preferred is a pharmaceutically pure state free of pyrogenic factors. Preferably, the isolated or purified casal-type serine protease inhibitors of the present invention are substantially free of other polypeptides.

The present invention relates to the use of such inhibitors described herein in medicine; And also the use of such inhibitors in the manufacture of a medicament. Thus, according to another aspect of the present invention, the pharmaceutical formulation comprises an inhibitor which is suitable for inhibiting activation of factor XII or activity of factor XIIa and preventing the formation and / or stabilization of three-dimensional arterial or venous thrombi. Pharmaceutical formulations are provided.

Therapeutic polypeptides described herein can be formulated into pharmaceutical formulations for therapeutic use. Purified protein may be dissolved in a conventional physiologically compatible aqueous buffer solution, optionally added with a pharmaceutical excipient to provide a pharmaceutical formulation.

Such pharmaceutical carriers and excipients, and suitable pharmaceutical formulations are well known in the art. See, eg, "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & Francis (2000) or " Handbook of Pharmaceutical Excipients ", 3 ^rd edition, Kibbe et al., Pharmaceutical Press (2000). In particular, pharmaceutical compositions comprising the polypeptides of the invention may be formulated in lyophilized or stable soluble form. The polypeptide may be lyophilized by various processes known in the art. Lyophilized formulations are reconstituted prior to use by adding one or more pharmaceutically acceptable diluents, such as sterile water for injection or sterile physiological salt solution.

The formulation of the composition is delivered to the subject by certain pharmaceutically suitable means of administration. Various delivery systems are known and can be used to administer the composition by any conventional route. Preferably, the composition of the present invention is administered systemically. For systemic use, the therapeutic proteins of the invention are for parenteral use (eg intravenous, subcutaneous, intramuscular, intraperitoneal, intracranial, pulmonary, intranasal or transdermal) or enteric (eg oral). Formulated according to conventional methods for delivery) (vaginal or vaginal or rectal). The most preferred route of administration is intravenous administration. The formulations may be administered continuously by infusion or bolus injection. Some formulations include a sustained release system.

Tablets and capsules for oral administration may contain conventional excipients such as binders, fillers, lubricants and wetting agents and the like. Oral liquid formulations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, and the like, or may be present as an anhydrous product for reconstitution with water or other suitable vehicle in use. Such liquid formulations may contain conventional additives such as suspending agents, emulsifiers, non-aqueous vehicles and preservatives.

Formulations suitable for topical application may be in the form of aqueous or oily suspensions, solvents, emulsions, gels or preferably emulsion ointments. Formulations useful for spray application may be in the form of sprayable liquids or dry powders.

The casal-type serine protease inhibitor polypeptide of the invention is administered to a patient sufficient to produce the desired effect while preventing or reducing the severity or spread of the condition or indication being treated without reaching a dosage that produces an unacceptable side effect. A therapeutically effective amount means a quantity. The exact dosage depends on many factors such as, for example, prescription, formulation and type of administration, and should be measured in preclinical and clinical trials for each individual indication.

The pharmaceutical composition of the present invention may be administered alone or in combination with other therapeutic agents. These agents may be incorporated as part of the same medicament.

Various products of the present invention are useful as medicaments. Accordingly, the present invention relates to a pharmaceutical composition comprising a casal-type serine protease inhibitor polypeptide, a polynucleotide of the present invention or a plasmid or vector of the present invention as described herein.

Modified DNA of the invention can also be incorporated into delivery vectors for use in human gene therapy.

The features, advantages and further features of the present invention will become apparent upon consideration of the accompanying drawings described below in conjunction with the following detailed description of the experiments performed and their results.

Figure 1: Model of pathological thrombosis discussed by Colman (Colman RW. 2006. Are hemostasis and thrombosis two sides of the same coin? J. Exp. Med. 203: 493-495).

Figure 2: Amino acid sequence similarity between Infestin-4 (I4) and SPINK-1 (SP). *, Identical amino acids; |, Similar amino acids.

Figure 3: The contact site of the R. prolytic susceptor and thrombin is represented by # and the contact site of SPINK-1 and chymotrypsin is represented by +.

4: amino acid sequence of infestin-4, SPINK1 and three SPINK1 mutants (K1-K3); With respect to the infestin-4 sequence, * represents the same amino acid; | Represents a similar amino acid. The underlined sequence of I4 was used to replace the 15 amino acids of SPINK-1 to produce mutant K1. Mutants K2 and K3 were generated by additional point mutations (underlined amino acids) on the K1 sequence.

Figure 5: Effect of rHA-infestin-4 on aPTT and FXII activity in mouse plasma in vitro

Figure 6: Prolongation of aPTT after administration of 100 and 200 mg / kg of rHA-infestin-4 (intravenous) up to 4.5 h (prior to and before administration) in mice

Figure 7: Inhibition of FXII after administration of 100 and 200 mg / kg of rHA-infestin-4 (intravenous) up to 4.5 h (prior to and before administration) in mice

8: rHA-infestin-4 (mean; n = 1-2 / time point) in mouse plasma over time after intravenous injection of 100 mg / kg

9: Comparison of pharmacokinetics of (His) 6-infestin and rHA-infestin-4 in mice

10: Effect of rHA-infestin-4 on hemostatic time (n = 10-15 / group, mean ± SD)

11: Effect of rHA-infestin-4 on total blood loss (n = 10-15 / group, mean ± SD)

12: Effect of rHA-infestin-4 on hemostatic time (n = 10-15 / group, individual data)

13: Effect of rHA-infestin-4 on total blood loss (n = 10-15 / group, individual data)

Example  One : Infestin -4, SPINK -1 and modified Kajal Of -type serine protease inhibitors Cloning

The SPINK-1 amino acid sequence was back translated into cDNA sequences containing optimized restriction sites optimized for mammalian cell expression. Dividing the nucleotide sequence of the resulting SPINK molecule comprising the native SPINK-1 signal peptide (see Figure 4) into three fragments, each of them oligonucleotides (Medigenomix, Martinsrid, Germany) Overlapping and custom synthesis. Each of the two variants of fragments 2 and 3 was generated and assembled in the following manner:

Produces SPINK-1 wild type with S1 + S2wt + S3wt

Create SPINK-K1 with S1 + S2K1 + S3wt

Create SPINK-K3 with S1 + S2K1 + S3K3

The nucleotide sequences of the fragments are provided by SEQ ID NOs: 7-11 (S1, SEQ ID NO: 7; S2wt, SEQ ID NO: 8; S2K1, SEQ ID NO: 9; S3wt, SEQ ID NO: 10; S3K3, SEQ ID NO: 11).

Assembly of the fragments was carried out as follows. Fragments obtained from Medigenomics in the cloning vector pCR2.1 (Invitrogen) were obtained from restriction endonucleases EcoRI / NarI (S1), NarI / KpnI (S2wt and S2K1) and KpnI / BamH1 (S3wt and S3K3), respectively. Cleaved, isolated from agarose gel and into EcoRI / BamH1 digested expression vector pIRESpuro3 [BD Biosciences].

a) S1 EcoRI / NarI + S2wt NarI / KpnI + S3wt KpnI / BamH1

b) S1 EcoRI / NarI + S2K1 NarI / KpnI + S3wt KpnI / BamH1

c) S1 EcoRI / NarI + S2K1 NarI / KpnI + S3K3 KpnI / BamH1

In combination to obtain plasmids p1171 (a), p1172 (b) and p1174 (c), respectively.

To generate the SPINK-K2 sequence, plasmid p1174 was prepared using oligonucleotides We2450 and We2451 (SEQ ID NOs: 12 and 13) with a commercially available mutagenesis kit (QuickChange XL Site Directed Mutagenesis Kit, Stratagene). Site directed mutagenesis reactions were performed according to the protocol. The resulting plasmid was named p1173.

C-terminal portion (fragment 14C, SEQ ID NO: 14), which was custom synthesized by overlapping the infestin-4 sequence with p1174 (N-terminal portion of SPINK-K3) and oligonucleotide (Medigenomeics, Martins Reid, Germany) Assembled from the coding sequence for). First, EcoRI / BamH1 fragments containing the coding sequence for the SPINK-K3 N-terminus were isolated from p1174 and cloned into EcoRI / BamH1 linearized plRESpuro3. The resulting plasmid was subsequently digested with BamH1 and NotI and a BglII / NotI fragment isolated from the pCR2.1 vector (provided from Medigenomics) containing the coding sequence for the 14C fragment was inserted. The resulting plasmid was called p1288 which contained the coding sequence for Infestin-4.

An expression vector with a hexahistidine tag attached to infestin-4 was constructed for purification purposes. For this purpose, using the commercially available mutagenesis kit (QuickChange XL Site Directed Mutagenesis Kit, Stratagen), p1288 as a template and oligonucleotides We2973 and We2974 as mutagenesis primers under the conditions described by the kit manufacturer SEQ ID NOs: 26 and 27). The resulting plasmid was named p1481, which encodes the 8 amino acid glycine / serine linker and 6 long histidine residues in the C-terminal extended Infestin-4 sequence (SEQ ID NO: 28).

Example 2: Cloning of Albumin Fusion Constructs

First, oligonucleotide We2467 was cloned into a commercial Mutagenesis kit (QuickChange XL Site Directed Mutagenesis Kit, Stratagen), which was cloned into the EcoRI site of pIRE-Spuro3 (BD Biosciences). And mutagenesis with site directed mutagenesis using We2468 (SEQ ID NOS: 15 and 16) to remove the stop codon and to remove the first portion of the glycine / serine linker and the BamH1 restriction site for insertion of the SPINK and Infestin-4 sequences. Introduced. The resulting plasmid was named p1192. Oligonucleotides We2470 introducing the BamH1 site at the 5'-end and the NotI site at the 3'-end as primers with the rest of the p1171, p1172, p1173, and p1174 and glycine / serine linkers as a template using the SPINK coding sequence (without signal peptide) And PCR using We2473 (SEQ ID NOs: 17 and 18). PCR fragments were digested with BamH1 / NotI, purified and inserted into p1192, and also cleaved with BamH1 / NotI. The resulting albumin fusion plasmid p1187 contained SPINK-1 wild type fused to albumin, p1188 SPINK-K1 fused to albumin, p1189 SPINK-K2 fused to albumin, and p1190 SPINK-K3 fused to albumin. Similarly, infestin-4 albumin expression plasmids were constructed but instead primers We2473 and We2623 (SEQ ID NOs: 18 and 19) were used on p1288. The resulting expression plasmid was named p1290. The amino acid sequences of the encoded proteins are provided as SEQ ID NOs: 20, 21, 22, 23 and 24, respectively.

Example 3: Transfection and Expression of Infestin-4 and Infestin-4 with SPINK Albumin Fusion Protein in Mammalian Cell Culture

Expression plasmids were grown in E. coli TOP10 (Invitrogen) and purified using standard protocol (Source: Qiagen). HEK-293 cells were transfected with Lipofectamine 2000 reagent (Invitrogen) and in serum-free medium [Invitrogen 293 Express] in the presence of 4 μg / ml puromycin. Grown. The transfected cell population was diffused through a T-flask through a roller bottle or small-scale fermenter in which the supernatant was harvested for purification. The expression yield in HEK-293 cells was 6-15 μg / mL for albumin fusion protein and about 0.5-1 μg / mL for His-tagged Infestin-4.

Example 4 Expression of His-tagged Infestin-4 and Infestin-4 Albumin Fusion Proteins in Yeast

The coding sequences of His-tagged Infestin-4 and Infestin-4 albumin fusion proteins were obtained from S. cerevisiae as described by Invitrogen, MoBiTec or Novozymes Biopharma. ( S. cerevisiae ) were transferred into an expression vector suitable for expression. As assessed from SDS PAGE analysis after Coomassie staining with expression in shake flask culture using standard growth medium, it was 30 and 50 μg / mL for albumin fusion protein and about 1 for His-tagged Infestin-4. Expression yields of from 5 μg / mL were obtained.

Example  5: Kajal  Purification of Inhibitor-Albumin Fusion Proteins

25 L of 0.2 μm filtered cell culture supernatant was concentrated to 1 L by ultrafiltration (10 kDa exclusion size), then dialyzed against 40 mM Tris / HCl pH 7.5 and again 0.2 μm filtered. The crude concentrate was further purified by PO anion exchange chromatography to POROS 50 PI (26 × 750). The column was equilibrated with 40 mM Tris / HCl pH 7.5. A washing step was performed after loading 15 column volumes (CV). The product was eluted with 40 mM Tris / HCl 1200 mM sodium chloride pH 7.5 in a linear gradient over 35 CV. Fusion proteins containing fragments were mixed and concentrated by ultrafiltration. Ultrafiltration on physiological sodium chloride solution gave a product of about 90% purity at a concentration of about 15 mg / mL.

Purification and detection of His-tagged Infestin-4 is commercially available kit (eg His-Tag purification and detection kit) containing Ni-NTA resin for purification and PentaHis antibody for detection of His-tagged protein. Manufacturer: Qiagen, Hilden, Germany).

Example  6: Kajal  Biochemical Properties of Inhibitor-Albumin Fusion Proteins

Measurement of identity / purity

Protein identity / purity was determined using SDS-PAGE (8-16%) using the standard procedure (NOVEX). Staining was performed with Coomassie Blue.

Protein concentration:

Protein concentration of albumin fusion proteins was determined using albumin specific ELISA, which is a basic practice known to those skilled in the art. In summary, microplates were incubated overnight at ambient temperature with 120 μL per well of capture antibody (Rabbit anti human albumin IgG, DAKO A0001) diluted 1: 14000 in Buffer A (Sigma C-3041). After another three washes with Buffer B (Sigma T-9039), each well was incubated with 200 μL of Buffer C (Sigma T-8793) for 1 hour at ambient temperature. After an additional three wash steps with Buffer B, serial dilutions of test samples in Buffer B and N Protein Standard SL in Buffer B (volume per well: 100 μL) were made from Dade Behring, 0.5-100 ng /. mL] was incubated at ambient temperature for 1 hour. After three wash steps with Buffer B, 100 μL of a 1: 12500 dilution of the detection antibody (Rabbit anti human albumin, DAKO P0356, labeled peroxidase) in Buffer B was added to each well and another 1 hour at ambient temperature Incubated. After three wash steps with Buffer B, 100 μL of substrate solution (TMB, Dade Bering, OUVF) was added per well and incubated in the dark at ambient temperature for 30 minutes. Sample was prepared for reading in a suitable microplate reader at 450 nm wavelength by addition of 100 μL of termination solution (Dade Bering, OSFA). The concentration of the test sample was then calculated using a standard curve using N protein standards as reference.

Active part Thromboplastin  Measurement of time

Activated partial thromboplastin time was measured in standard human plasma (SHP, Dadebering), where different amounts of each inhibitor were added to the imidazole buffer in a total volume of 200 μL. 50 μL of this solution was added to 50 μL of Patrometin SL (Dade Bering) and incubated at 37 ° C. for 120 seconds. Subsequently, 50 μL of calcium chloride solution (25 mM) was added to initiate the reaction.

The procedure was carried out on BCT (Bering Coagulation Timer) according to the conditions suggested by the manufacturer.

Prothrombin  Measure of time:

Prothrombin time was measured in standard human plasma (Dade Bering), and the activating reagent was Tromborel S (Dade Bering). 100 μl of Tromborel S was added to 50 μL of sample (see above) after 15 seconds of incubation time. The procedure was carried out on BCT (Bering Coagulation Timer) according to the conditions suggested by the manufacturer.

result:

These tests demonstrate that Kazal-type inhibitors can inhibit the endogenous pathway with little effect on the exogenous pathway expressed by nearly constant PT.

Example 7: Highly Effective in Preventing Vascular Occlusion in a Mouse Model for Arterial Thrombosis Infestin -4 albumin Fusion

In vitro in vitro spiking experiments were performed to evaluate the dose required to strongly protect mice from arterial thrombosis. Spiking rHA-infestin-4 into mouse plasma resulted in decreased FXII activity and prolongation of aPTT, but the PT remained virtually unchanged.

Since very clear inhibition was observed after spikes of mouse plasma in vitro, mice were treated intravenously with rHA-infestin-4 and the aPTT and FXII activities were evaluated over time (FIGS. 6 and 7). In addition, plasma levels of rHA-infestin-4 were measured at the time points indicated in Table 5.

The test indicated that a single intravenous injection of 400 mg / kg of rHA-infestin-4 prolonged aPTT and decreased FXII activity for at least 1 hour. Thus, a single injection could protect mice from thrombotic vascular occlusion in the FeCl ₃ model of thrombosis. Therefore, animals were administered intravenously with 400 mg / kg of rHA-infestin-4 and the vascular occlusion rate and time to occlusion were measured.

Animals received one intravenous injection of rHA-infestin-4 at a dose of up to 400 mg / kg at t = 0. To assess arterial thrombosis, the abdominal artery was exposed during deep anesthesia. Basic blood flow was measured by placing an ultrasonic flow probe around the vessel. To initiate thrombosis, a 0.5 mm ² patch filter paper saturated with 10% ferric chloride solution was placed underneath the abdominal artery of the flow probe. After a 3-minute exposure period, filter paper was removed and blood flow was monitored for 60 minutes to determine the incidence of thrombotic occlusion.

Table 6 shows that 82% of vehicle treated animals showed thrombosis. In contrast, none of the 10 mice treated with rHA-Infestin-4 developed thrombosis. This effect is dose-dependent and is the opposite of decreasing incidence of occlusion, time until incidence of occlusion increases.

FXII k.o. Animals are similarly protected from thrombosis, but at the same time no hemostatic deficiency is observed, so hemostasis was analyzed in a similar manner in mice intravenously treated with rHA-infestin-4 of 400 mg / kg or less. For this purpose, animals were anesthetized by using intravenous once injection of about 60 mg / kg with narcorene. rHA-Infestin-4 was injected at the same time point 15 minutes before the lesion of the animal, ie at the same dose as in the experiment to evaluate the antithrombotic effect.

Hemostasis was quantified by measuring blood loss at the end of the 30 minute observation period with a sensor for time to hemostasis and hemostasis. The volume of total blood loss was calculated by measuring the HGB present in the saline used for immersion of the tail. Therefore, HGB of animals was considered. The tail was removed by about 3 mm by using a small knife under deep anesthesia. At the same time as the lesion, the tails were immersed in saline which was pre-warmed and kept at the physiological body temperature of the mice using a water bath during the observation period. Observation time to monitor bleeding was 30 minutes. All test products were administered intravenously 15 minutes before the start of the observation period (tail cut).

All major parameters for hemostasis within the observation period, time to hemostasis and blood loss did not show a clear difference between the two treatment groups and the vehicle control group (Tables 7, 8, Figures 10-13).

Only Infestin-4, i.e., Infestin-4 that is not fused with albumin (see, eg, His-tagged Infestin-4) is tested for potential protection from thrombosis. A dose of rHA-infestin-4, approximately equivalent to 400 mg / kg, is injected intravenously once into the mouse 15 minutes before thrombosis induction. Induction and evaluation of thrombosis is performed in the same manner as described for rHA-infestin-4. Infestin-4 is applied by continuous infusion or repeated injections to overcome rapid elimination, which is a standard procedure that rapidly removes from the circulation and maintains high plasma levels of compounds that lose activity due to mechanisms other than pharmacokinetic reasons. .

The results showed that the group of rHA-infestin-4 treated mice showed FXII k.o. No risk of thrombosis or bleeding consistent with the results of the mouse group (also shown).

Example  8: arterial thrombosis Rat FeCl ₃  Marketed in the model FXII (a) inhibitors Berinert ^® P is aPTT , PT  And effects on vascular occlusion

A number of in vitro and in vivo tests to evaluate the suitability of FXII (a) FXII (a) as marketed in the efficacy of inhibiting inhibitors Berinert ^® P (C1 esterase inhibitor) was performed.

The test goal was to measure the effects on aPTT and PT as well as FXII activity in rat plasma and to assess potential anti-thrombotic effects in the rat FeCl ₃ model of arterial thrombosis.

Rats were anesthetized and blood samples were collected from the posterior orbit and processed into plasma for measurement of factor XII activity according to standard procedures. These plasma samples were spiked with Berinert ^ⓒ P and tested directly for FXII activity.

Berinert ^ⓒ P spikes were tested for their effect on FXII activity in vitro. Substantial FXII inhibition was observed at high concentrations (Table 9).

As significant inhibition of FXII was achieved, an in vivo test was performed. The potential prophylaxis of thrombosis was measured by intravenously administering rats at a dose of 1200 U / kg using Bererinert ^ⓒ P. Dosage was selected to achieve a plasma concentration of 10-15 U / mL. For evaluation of arterial thrombosis, the carotid artery and jugular vein were exposed under deep anesthesia. The cannula was intubated intravenously for drug administration. To monitor blood flow, ultrasonic flow probes were placed around the carotid artery. To initiate thrombosis, a 2.5 mm ² patch filter paper saturated with 35% ferric chloride solution was placed underneath the carotid artery of the flow probe. After 3 minutes exposure, filter paper was removed and blood flow was monitored for 60 minutes to determine the incidence of thrombotic occlusion. APTT, PT and FXII activity was measured at the end of the observation period.

Three minutes treatment of the carotid artery with 35% ferric chloride resulted in a thrombotic obstruction at 100% rate (Table 10). Although high doses of Berinert ^® P resulted in increased aPTT and moderate FXII inhibition, no positive effect on occlusion rate was observed.

summary:

High concentrations of Berinert ^® P significantly inhibited FXII in vitro. However, high doses of Berinert ^® P were also insufficient in the FeCl ₃ model of arterial thrombosis. This dose of Berinert ^® P was close to technical limitations because the application of higher doses resulted in higher protein loads and non-physiological injection volumes.

Example  9: on the mouse ( His ) 6- Infestin  And rHA - Infestin Comparison of Pharmacokinetics of -4

His-tagged Infestin-4 ((His) 6-infestin-4) or Infestin-4 albumin fusion (rHA-infestin-4) formulations were administered intravenously to 28 NMRI mice. The dose was (20 mg / kg) body weight for (His) 6-infestin-4 and 200 mg / kg body weight for rHA-infestin-4. This dosage corresponds to an amount equivalent to the active ingredient of the two proteins, Infestin-4.

Blood samples were collected at appropriate intervals from 5 minutes after application of the test substance. Infestin-4 antigen content was continuously quantified by an ELISA assay specific for infestin-4. The mean value of the treatment group was used for the calculation. The half-life for each of the protein expression t _1/2 = ln2 _/ k is calculated by using the time on the removal according to (here, k is the slope of the regression line) beta. The results are shown in Table 9 (n = 1-4 / time point; average).

The final half life calculated for rHA-infestin-4 is 3 hours, while the final half life calculated for (His) 6-infestin-4 is 0.3 hours. Thus, the apparent increase in final half-life is 10-fold for rHA-infestin-4 compared to (His) 6-infestin-4.

<110> CSL Behring GmbH <120> Therapeutic application of Kazal-type serine protease inhibitors <130> 2007_M001_A119 <150> EP 07002903.8 <151> 2007-02-12 <160> 25 <170> KopatentIn 1.71 <210> 1 <211> 56 <212> PRT <213> Homo sapiens <400> 1 Asp Ser Leu Gly Arg Glu Ala Lys Cys Tyr Asn Glu Leu Asn Gly Cys 1 5 10 15 Thr Lys Ile Tyr Asp Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Pro             20 25 30 Asn Glu Cys Val Leu Cys Phe Glu Asn Arg Lys Arg Gln Thr Ser Ile         35 40 45 Leu Ile Gln Lys Ser Gly Pro Cys     50 55 <210> 2 <211> 53 <212> PRT <213> Artificial <220> <223> Artificial <400> 2 Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn 1 5 10 15 Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Pro Asn Glu Cys             20 25 30 Val Leu Cys Phe Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln         35 40 45 Lys Ser Gly Pro Cys     50 <210> 3 <211> 53 <212> PRT <213> Artificial <220> <223> Artificial <400> 3 Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn 1 5 10 15 Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Gly Asn Glu Cys             20 25 30 Met Leu Cys Ala Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln         35 40 45 Lys Glu Gly Pro Cys     50 <210> 4 <211> 54 <212> PRT <213> Artificial <220> <223> Artificial <400> 4 Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn 1 5 10 15 Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Gly Asn Glu Cys             20 25 30 Met Leu Asn Cys Ala Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu Ile         35 40 45 Gln Lys Glu Gly Pro Cys     50 <210> 5 <211> 48 <212> PRT <213> Triatoma infestans <400> 5 Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn Tyr Val Pro Val Cys 1 5 10 15 Gly Ser Asp Gly Lys Thr Tyr Gly Asn Pro Cys Met Leu Asn Cys Ala             20 25 30 Ala Gln Thr Lys Val Pro Gly Leu Lys Leu Val His Glu Gly Arg Cys         35 40 45 <210> 6 <211> 585 <212> PRT <213> Homo sapiens <400> 6 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu             580 585 <210> 7 <211> 81 <212> DNA <213> Artificial <220> <223> Artificial <400> 7 gaattcgcca ccatgaaggt gaccggcatc ttcctgctgt ccgccctggc cctgctgtcc 60 ctgtccggca acaccggcgc c 81 <210> 8 <211> 84 <212> DNA <213> Artificial <220> <223> Artificial <400> 8 ggcgccgact ccctgggccg cgaggccaag tgctacaacg agctgaacgg ctgcaccaag 60 atctacgacc ccgtgtgcgg tacc 84 <210> 9 <211> 75 <212> DNA <213> Artificial <220> <223> Artificial <400> 9 ggcgccgact ccctgggccg cgaggtgcgc aacccctgcg cctgcttccg caactacgtg 60 cccgtgtgcg gtacc 75 <210> 10 <211> 105 <212> DNA <213> Artificial <220> <223> Artificial <400> 10 ggtaccgacg gcaacaccta ccccaacgag tgcgtgctgt gcttcgagaa ccgcaagcgc 60 cagacctcca tcctgatcca gaagtccggc ccctgctgag gatcc 105 <210> 11 <211> 108 <212> DNA <213> Artificial <220> <223> Artificial <400> 11 ggtaccgacg gcaacaccta cggcaacgag tgcatgctga actgcgccga gaaccgcaag 60 cgccagacct ccatcctgat ccagaaggag ggcccctgct gaggatcc 108 <210> 12 <211> 25 <212> DNA <213> Artificial <220> <223> Artificial <400> 12 cgagtgcatg ctgtgcgccg agaac 25 <210> 13 <211> 25 <212> DNA <213> Artificial <220> <223> Artificial <400> 13 gttctcggcg cacagcatgc actcg 25 <210> 14 <211> 107 <212> DNA <213> Artificial <220> <223> Artificial <400> 14 agatctgacg gaaagaccta cggcaacccc tgcatgctga actgtgccgc ccagaccaag 60 gtgcccgggc tcaagctggt gcacgagggc cgctgctagg cggccgc 107 <210> 15 <211> 37 <212> DNA <213> Artificial <220> <223> Artificial <400> 15 gctgccttag gcttaggagg atccagcgct gtgaagg 37 <210> 16 <211> 37 <212> DNA <213> Artificial <220> <223> Artificial <400> 16 ccttcacagc gctggatcct cctaagccta aggcagc 37 <210> 17 <211> 29 <212> DNA <213> Artificial <220> <223> Artificial <400> 17 cgcgcggccg cgatcctcag caggggccg 29 <210> 18 <211> 42 <212> DNA <213> Artificial <220> <223> Artificial <400> 18 gcgggatccg gggggagcgg cggctccgac tccctgggcc gc 42 <210> 19 <211> 18 <212> DNA <213> Artificial <220> <223> Artificial <400> 19 ctgctggcgg ccgcctag 18 <210> 20 <211> 650 <212> PRT <213> Homo sapiens <400> 20 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser Gly Gly Ser Gly             580 585 590 Gly Ser Asp Ser Leu Gly Arg Glu Ala Lys Cys Tyr Asn Glu Leu Asn         595 600 605 Gly Cys Thr Lys Ile Tyr Asp Pro Val Cys Gly Thr Asp Gly Asn Thr     610 615 620 Tyr Pro Asn Glu Cys Val Leu Cys Phe Glu Asn Arg Lys Arg Gln Thr 625 630 635 640 Ser Ile Leu Ile Gln Lys Ser Gly Pro Cys                 645 650 <210> 21 <211> 647 <212> PRT <213> Homo sapiens <400> 21 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser Gly Gly Ser Gly             580 585 590 Gly Ser Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe         595 600 605 Arg Asn Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Pro Asn     610 615 620 Glu Cys Val Leu Cys Phe Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu 625 630 635 640 Ile Gln Lys Ser Gly Pro Cys                 645 <210> 22 <211> 647 <212> PRT <213> Homo sapiens <400> 22 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser Gly Gly Ser Gly             580 585 590 Gly Ser Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe         595 600 605 Arg Asn Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Gly Asn     610 615 620 Glu Cys Met Leu Cys Ala Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu 625 630 635 640 Ile Gln Lys Glu Gly Pro Cys                 645 <210> 23 <211> 648 <212> PRT <213> Homo sapiens <400> 23 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser Gly Gly Ser Gly             580 585 590 Gly Ser Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe         595 600 605 Arg Asn Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Gly Asn     610 615 620 Glu Cys Met Leu Asn Cys Ala Glu Asn Arg Lys Arg Gln Thr Ser Ile 625 630 635 640 Leu Ile Gln Lys Glu Gly Pro Cys                 645 <210> 24 <211> 647 <212> PRT <213> Homo sapiens <400> 24 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln             20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu         35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys     50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Ser Gly Gly Ser Gly             580 585 590 Gly Ser Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe         595 600 605 Arg Asn Tyr Val Pro Val Cys Gly Ser Asp Gly Lys Thr Tyr Gly Asn     610 615 620 Pro Cys Met Leu Asn Cys Ala Ala Gln Thr Lys Val Pro Gly Leu Lys 625 630 635 640 Leu Val His Glu Gly Arg Cys                 645 <210> 25 <211> 294 <212> PRT <213> Homo sapiens <400> 25 Asp Ser Leu Gly Arg Glu Val Arg Asn Pro Cys Ala Cys Phe Arg Asn 1 5 10 15 Tyr Val Pro Val Cys Gly Thr Asp Gly Asn Thr Tyr Gly Asn Glu Cys             20 25 30 Met Leu Cys Ala Glu Asn Arg Lys Arg Gln Thr Ser Ile Leu Ile Gln         35 40 45 Lys Glu Gly Pro Cys Gly Gly Ser Gly Gly Ser Gly Gly Ser Glu Pro     50 55 60 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 65 70 75 80 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp                 85 90 95 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp             100 105 110 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly         115 120 125 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn     130 135 140 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 145 150 155 160 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro                 165 170 175 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu             180 185 190 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn         195 200 205 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile     210 215 220 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 225 230 235 240 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys                 245 250 255 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys             260 265 270 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu         275 280 285 Ser Leu Ser Pro Gly Lys     290  

Claims (18)

A mutant Kazal inhibitor derived from SPINK-1 that is mutated to increase homology to Infestin-4 and is suitable for preventing the formation and / or stabilization of three-dimensional arterial or venous thrombi. ). The mutant casal inhibitor of claim 1, wherein the site of contact with the inhibited protease is from domain 4 of the casal-type inhibitor infestin. 3. The mutant casal inhibitor of claim 2, wherein the additional amino acid position in the modified casal inhibitor sequence is from domain 4 of infestin. The mutant casal inhibitor according to any one of claims 1 to 3, which is SPINK K1, K2 and K3 (SEQ ID NOs: 2, 3 and 4). The mutant casal inhibitor and / or infestin or fragment thereof, preferably infestin 3-4 or more preferably infestin-, according to any of the preceding claims, linked to a half-life enhancing polypeptide. 4. 6. The half-life enhancing polypeptide according to any one of claims 1 to 5, wherein the half-life enhancing polypeptide is from a member of the human albumin protein family, i.e. albumin, alphamin, alpha-fetoprotein or vitamin D binding protein. Inhibitors. The inhibitor according to any one of claims 1 to 5, wherein the half-life enhancing polypeptide is human albumin or a variant or domain or part thereof. The inhibitor according to any one of claims 1 to 5, wherein the half-life enhancing polypeptide is an immunoglobulin or part thereof. 9. The inhibitor of claim 8, wherein the immunoglobulin moiety is an IgG Fc moiety. 10. The inhibitor according to claim 5, wherein the half-life enhancing polypeptide is linked to the casal inhibitor residues via a linker, preferably a cleavable linker. The inhibitor of claim 10, wherein the linker is cleavable by coagulation proteases of the endogenous coagulation pathway. 12. The inhibitor of claim 11, characterized by a linker cleavable by FXIIa. A medicament comprising an inhibitor according to any one of claims 1 to 12 or comprising infestin or a fragment thereof. An inhibitor according to any one of claims 1 to 12 and / or infestin or a fragment thereof for use as a medicament. Use of the inhibitor according to any one of claims 1 to 12 and / or infestin or a fragment thereof for the manufacture of a medicament for the treatment or prophylaxis of a condition or disease associated with arterial thrombus formation. Arterial thrombosis, stroke, myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and / or hypotonic shock, edema including hereditary angioedema, bacterial infection, arthritis, pancreatitis or joint gout, disseminated intravascular coagulation (DIC) and Use of an inhibitor according to any one of claims 1 to 12 and / or infestin or a fragment thereof for the manufacture of a medicament for the treatment or prophylaxis of a disease associated with pathological kinin formation such as sepsis. 13. The inhibitor and / or infestin or fragment thereof, preferably infestin 3-4 or infestin-4, according to any one of claims 1 to 12, is linked to arterial thrombus formation by A method of increasing the efficacy of said inhibitor in the treatment of a condition or disease. A method for producing an inhibitor and / or infestin or a fragment thereof according to any one of claims 1 to 12 via recombinant means in eukaryotic cells, bacteria, yeast, plant or insect cells or transgenic animals.

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